The indirect immunofluorescence assay (IFA) is one of the most used techniques in immunology to detect in a certain sample the presence of specific unknown antigens or antibodies whose known counterpart is variously linked to a fluorophore. The fluorophore is a fluorochrome, generally fluorescein, which absorbing ultraviolet rays emits a green light and therefore an observable light with an illuminated microscope.
The method of analysis foresees that in the research of the unknown antibody the sample, appropriately diluted with a phosphate buffer or a saline solution, is put in contact with known antigens and fixed on the window of a slide. The slide is thus characterized by an assembly of windows (dedicated areas) on which the analysis are done.
The substrate is therefore treated and prepared with appropriate known methods, in such a way as to generate a specific antigen. It is useful to remember here that the antigen is any element extraneous to the organism, capable of inducing an immunospecific response with the generation of a population of reactive lymphocytes B and consequent production of specific antibodies.
If a specific illness is researched, a specific substrate is arranged that represents a specific antigen capable of linking with the specific antibodies eventually present in the sample under exam, in case it is infected.
The operation of arrangement of the sample on the window must be done in a few minutes, otherwise a process of degeneration of the substrate starts and all the results risk to be distorted.
After arranging the sample on the window, and waiting for the necessary incubation time, an accurate wash with a buffer solution must be done and an anti-immunoglobulin antiserum must be added, of the species from which the serum under exam comes from, marked with fluorescein (FITC).
After waiting a further incubation time, the antiserum is washed again with a buffer solution.
At the end of this phase, on each window there is applied mounting medium to preserve the fluorescence from a quick decay. It is finally arranged on the slide a cover slide that has a thickness generally of about a tenth of a millimeter. The mounting medium is a solution of glycerol at 10% in PBS that is used to “crystallize” the situation obtained on the window and guarantee a better retention of the fluorescent light. At this point, the cover slide is applied that has a thickness generally of about a tenth of a millimeter, for example 0.2 mm. At the current state, it is applied by hand and moved with a suction pen. The cover slide intensifies the duration of the complex biochemicals obtained but must be applied with care to avoid the formation of bubbles that would affect the subsequent vision at the microscope.
At this point, the slide is ready for the analysis at the microscope through the use of objectives of 20.times. and 40.times. magnification and the presence of the antibody conjugated with the fluorophore thus becomes an evidence of the presence of the illness researched.
At the current state of the art, the necessary procedures described above are done totally or in part manually, the whole requiring the continuous presence of an operator and above all lengthening the procedure times. In particular, the operations of preparation of the slide can be manual and its subsequent transfer in the section of the microscope is manual. Not only does the whole lengthen the analysis times but it also risks to make exceed said limit times within which the slide must be arranged, avoiding deteriorations of the materials in analysis that would render the subsequent detections vane.
Currently, there exist automatic preparers (processors) that at the end of their preparation process provide slides that, following an operation of manual assembly (deposition on the slide of a fixing liquid and positioning of the cover slide), are ready for the reading and interpretation at the traditional microscope. A feature of these slides is the rapid decay of the fluorescent signal that is read at the microscope and therefore require a quick reading after their preparation and are not re-usable after a long time for a subsequent re-reading.
As for the reading at the microscope in the past, instead, a telecamera was mounted on the microscope and some fields of interest were photographed and saved in the computer. The choice of the fields was at the operator's discretion. In the last years automatic acquisition and interpretation systems have been introduced, which operate as follows: acquisition of a pre-set number of images per window in pre-defined positions with subsequent automatic discrimination of the positive/negative cases and recognition of the cellular pattern. In the cases in which the operator is not sure of the automatic evaluation done by the software, it is indispensable to re-evaluate the slide at the traditional microscope because the number of fields acquired is not sufficient to visualize the entire slide and complete the evaluation in each situation.
To date, ultimately and considering what has been said so far, the process of diagnostic analysis of biological samples in autoimmunity takes place according to the following steps:
The operator inserts the samples in the system of preparation of the slides;
The system prepares the slides automatically;
The operator takes off the slides and assembles them manually (adding “Mounting Medium” and cover slide);
The operator reads at the traditional microscope the slide or acquires some fields of the slide in the case in which an automatic reading system at the microscope is present.
It results evident that during the process the continuous presence of an operator is required and that after some days the slides are deteriorated and not readable anymore due to the loss of the fluorescent signal.
Publication WO2014/132094 is further known, which corresponds to the preamble of claim 1.
Such publication describes a machinery that allows to automatize a good part of the process of analysis of a slide.
The machinery foresees a loading station of the slides and a mobile system that prepares the slides with the various necessary reagents. The slide is then automatically moved under a unit of acquisition of the images where all the images at various magnifications are acquired, to be able to be then normally analyzed also from remote.
The machinery described in such publication, even if being able to automatize the process, is not exempt from some technical inconveniences, in particular in the field of auto-immunity.
The process of preparation of the slide and analysis is not in fact totally automatized, requiring the presence of an operator at least during all the preparatory phase of the slide.
In particular, it is known that is it necessary a pre-dilution phase of the samples that can require a variable time, sometimes also some hours, for its completion. In that sense, the operation of loading of the slides in the machine must take place, through an operator, only after the operator has done the dilution of samples and such operation has been completed.
In that sense, in accordance with said publication and with the known machines in general that operate in the field of auto-immunity, at the current state it is impossible to arrange contextually the slides in the machine and start a pre-dilution phase of the samples, since the time within which the deterioration of the substrate initiates due to the moisture of the air can be of about 15-30 minutes, while the operation of pre-dilution can require longer times, also a couple of hours.
In a preliminary phase, therefore, the presence of an operator is necessary that first starts the pre-dilution of the liquids, waits for the completion of such operation and then loads the slides on the machinery. All this, actually, evidently renders the machine automatic only in part.